Off-target silencing is a major concern when using RNA interference (RNAi). Conventional 19+2 siRNA structures can undergo imperfect pairing of the antisense strand with mRNA targets or incorporation of the sense strand into RISC complexes, which results in unintended cleavage of targets. Thus, such conventional 19+2 siRNA structures show considerable non-specific silencing (Jackson, A. L. & Linsley, P. S., Trends Genet., 20(11): 521, 2004). A recent study conducted by the present inventor has reported an asymmetric siRNA (16+3A structure) having a shortened sense strand (Chang C. I. et al., Mol. Ther., 17(4): 725, 2009). This asiRNA structure overcomes the problems associated with the 19+2 siRNA structure, such as sense strand-mediated silencing and RNAi machinery saturation. Many other researchers have also reported various siRNA modifications that reduce sense-strand-mediated non-specific silencing (Elmen, J., et al., Nucleic Acids Res., 33(1): 439, 2005; Sano, M., et al., Nucleic Acids Res., 36(18): 5812, 2008; Sun, X., et al., Nat. Biotechnol., 26(12): 1379, 2008). However, as compared to such studies, studies on the reduction in antisense-mediated off-target silencing were extremely small.
Jackson et al. first demonstrated that siRNA-mediated gene silencing is sequence-dependent rather than target-dependent (Jackson, A. L., et al., Nat. Biotechnol., 21(6): 635, 2003; Jackson, A. L., et al., Rna, 12(7): 1179, 2006b). Additionally, initiation of target cleavage by RISC is caused by limited complementarity between RNA double strands and transcripts. Birmingham et al. demonstrated that unintended pairing of the 3′ UTR of transcripts with the seed region of siRNA is the major cause of off-target effects (Birmingham et al., Nat. Methods, 3(3): 199, 2006). Pairing between the hexamer seed region of an siRNA guide strand (e.g., nucleotides 2-7) and complementary sequences in the 3′ UTR of mature transcripts is a primary determinant of off-target gene regulation (Lin et al., Nucleic Acids Res., 33(14): 4527, 2005; Anderson et al., Rna, 14(5): 853, 2008). Such off-targets can induce a measurable amount of phenotypic changes that can account for up to 30% of the positive hits in RNAi based phenotypic screens. In view of widespread off-target silencing mediated by conventional siRNAs having a 19+2 structure, any chemical or structural modification of the siRNA backbone, which can reduce off-target silencing while maintaining intended silencing efficiency, is a great concern.
A collaborative study between Dharmacon Research (Lafayette, Colo.) and Rosetta Inpharmatics (Seattle, Wash.) demonstrated that methyl-groups added to the 2′ position of the ribosyl ring of the nucleotide at a specific position in the siRNA guide strand significantly reduced siRNA-mediated off-target effects (Jackson et al., Rna, 12(7): 1197, 2006). Additionally, it was found that chemical modification of the base at position 2 in the guide strand was most effective in reducing both the number and extent of off-target effects without significantly influencing the silencing of an intended target. However, Ambion Inc. compared a 2′OMe-modified siRNA with an LNA-modified siRNA and found that the LNA-modified siRNA was superior in reducing off-target effects mediated by the antisense strand (Puri et al., Nucleic Acids Symp. Ser. 2008). However, it was shown that such chemical modifications successfully reduced the antisense off-target effects of siRNA, while some among them, such as 2′-OMe, could also reduce on-target silencing efficiency. Furthermore, such chemical modification strategies could not be applied to siRNAs which are expressed in cells.
Accordingly, the present inventors have made extensive efforts to provide a novel siRNA structure, which has high gene silencing efficiency while minimizing off-target effects caused by the antisense strand, and as a result, have found that a novel siRNA molecular structure constructed by the present inventors has excellent gene silencing effects without showing off-target effects caused by the antisense strand, and thus has improved target selectivity, thereby completing the present invention.